ProjectSummary Clostridium difficile infection (CDI) is the most common cause of antibiotic-associated diarrhea and gastroenteritis-associated death among developed countries, and it is classified as one of thetopthree?urgentthreats?bytheUSCentersforDiseaseControlandPrevention(CDC).The diseaseassociatedwithCDIismainlymediatedbytwohomologousexotoxins,TcdAandTcdB. Theytargetanddisruptthecolonicepithelium,leadingtodiarrheaandcolitis.Receptor-binding is a critical step in the toxin?s action and largely determine toxin tropisms, and these receptors are also molecular targets for therapeutic interventions. The goal of our proposal is to identify and characterize toxin receptors, establish a structural basis for toxin-receptor recognition, and harness this knowledge to gain a mechanistic understanding of the biology, pathogenesis, and therapy of TcdA and TcdB. We will take a multi-disciplinary approach, bringing together the expertise in toxin structure/function of Dr. Rongsheng Jin?s group with the expertise in toxin receptor identification/pathogenesis of Dr. Min Dong?s group. This proposed work is built on bothourpublishedworkonidentifyingFrizzled(FZD)proteinsasTcdBreceptors,andextensive preliminary data on toxin-receptor interactions. We will focus on three aims: (1) elucidate the structuralbasisandpathogenicrelevanceofTcdB-FZDrecognition;?(2)understandthestructure of TcdB and the influence of FZD binding;? and (3) identify and characterize novel TcdA receptors through a CRISPR/Cas9 mediated genome-wide screens. These proposed studies will provide a comprehensive understanding of toxin-receptor interactions and how they contributetothetoxinbiologyandpathogenesisofTcdAandTcdB.